Biomedical subjects

N Navaratnam

Publications and source records attributed to N Navaratnam.

31 records · Page 2Linked to original sources

PubMed1993

Apolipoprotein B mRNA editing is associated with UV crosslinking of proteins to the editing site.

Apolipoprotein (apo) B100 mRNA undergoes editing of C-6666 to a U residue, which generates a stop-translation codon and defines the carboxyl terminus of apoB48. To aid purification of the editing enzyme we have undertaken UV crosslinking of a 32P-labeled substrate for apoB mRNA editing in vitro to proteins in an enterocyte editing extract. Proteins of 60 (p60) and 43 (p43) kDa, prominent among crosslinking bands, were competed for by unlabeled substrate, but not by nonspecific RNA, and did not crosslink to antisense RNA. Editing in vitro and UV crosslinking were inhibited by NaCl and vanadyl ribonucleoside complexes and by chemical modification of sulfhydryl, imidazolium, and guanidinium groups on the protein. The editing activity copurified predominantly with p60. To define the binding site for p60 on the substrate RNA, a series of scanning and point mutant RNAs, previously used to define nucleotides 6671-6681 as essential for editing, were used in competition studies with wild-type substrate. Results demonstrated that p60 binding is centered on nucleotides 6671-6674. We suggest that p60 contains the RNA-recognition component of the apoB mRNA-editing enzyme.

Animals↗

PubMed1992

Endorphin mediation of mesenteric blood flow after endotoxemia in sheep.

BACKGROUND AND METHODS: The administration of endotoxin in small dosages to sheep results in an acute decrease followed by an increase in cardiac output. It has previously been determined that the initial decrease in output was the result of a reduction in blood flow to the mesenteric areas. These changes were associated with increased blood concentrations of beta endorphin. The present study was accomplished to determine if the systemic cardiovascular response to endotoxin could be affected by the administration of an opiate receptor-blocking agent. Female range sheep (n = 12) were prepared for chronic study by implantation of cardiopulmonary catheters and a flow probe on the cephalic mesenteric artery. Endotoxin (Escherichia coli, 1 microgram/kg) was administered to these animals. Half of the sheep were treated with naloxone (2 mg/kg + 2 mg/kg.hr), and the remainder with an equivalent volume of sodium chloride (0.9%). RESULTS: In untreated sheep, cardiac indices decreased by 15% to 20% (5.1 +/- 0.1 to 4.2 +/- 0.4 L/min.m2) between 0 and 1 hr and 2 and 5 hrs after endotoxin (4.5 +/- 0.2 L/min.m2), and then increased to a value 40% (7.2 +/- 0.6 L/min.m2) above baseline by 12 hrs. Flow in the cephalic mesenteric artery decreased in a pattern similar to the reduction in cardiac index (962 +/- 152 [time, T = 0] to 379 +/- 111 [T = 0.8] and 384 +/- 88 mL/min [T = 4.0], p less than .05) but did not increase to the same extent (1008 +/- 153 mL/min [T = 4.0], p greater than .05). There was a concomitant increase in the plasma beta-endorphin concentrations as the blood flow decreased (5 +/- 4 [T = 0] to 40 +/- 15 pg/mL [T = 0.8; untreated group], p less than .05; and 10 +/- 4 to 50 +/- 7 pg/mL [T = 0.8; naloxone-treated group], p less than .05). In the naloxone-treated group, the same pattern of cardiac output change was noted with endotoxin; however, reduction of mesenteric artery flow was only 30% (1118 +/- 129 to 908 +/- 122 mL/min, p less than .05) of the value seen in the untreated animals (962 +/- 152 to 379 +/- 111 mL/min, p less than .05). These changes in mesenteric blood flow were statistically significant from one another. As the cardiac output increased in the sheep treated with the opiate antagonist, mesenteric blood flows increased 20% above the baseline value (1391 +/- 199 mL/min, p less than .05). CONCLUSIONS: The decrease in cardiac output noted with endotoxin can be accounted for by the decrease in the blood flow in the cephalic mesenteric artery. This phenomenon can be attributed, at least in part, to the release of endorphins. There is both a vasodilation and constriction during endotoxemia in the ovine model.

Animals↗

PubMed1992

Immediate positive pressure ventilation with positive end-expiratory pressure (PEEP) improves survival in ovine smoke inhalation injury.

BACKGROUND: The purpose of this study was to compare the effects of immediate initiation of positive pressure ventilation (PPV) with positive end-expiratory pressure (PEEP) versus the initiation of PPV with PEEP only after hypoxemia ensued following severe smoke inhalation injury. METHODS: We prospectively evaluated chronically instrumented adult sheep treated with immediate versus delayed PPV with PEEP and compared oxygen requirements, hemodynamics, pleural fluid formation, postinjury survival, and tracheobronchial pathologic processes among groups. The immediate group (group I; smoke, n = 6; sham, n = 2) underwent tracheostomy and bilateral chest tube placement before they received inhalation injury. They were then immediately placed on PPV with PEEP (12 cm H2O). The animals in the delayed group (group D) (n = 6) were placed on PPV with PEEP when arterial hypoxemia (PaO2 < 80 mm Hg [11.2 kPa] on 0.4 FIO2) or respiratory distress developed. RESULTS: Groups were matched for smoke exposure and peak carboxyhemoglobin. Both groups developed a characteristic decrease in PaO2/FIO2 ratio. Initiation of PPV + PEEP improved PaO2 in the delayed group (69 +/- 7 to 126 +/- 21 mm Hg [9.2 +/- 0.9 to 16.7 +/- 2.8 kPa]). Pleural fluid output was greater in the immediate group compared with the delayed group (1559 +/- 415 vs. 426 +/- 236 mL). At 96 hours after injury five of six animals in the delayed group had died. In contrast, six of six animals in the immediate smoke group survived 96 hours (p < 0.05 versus delayed group). The immediate group had fewer and less extensive tracheobronchial casts at necropsy. CONCLUSIONS: Immediate PPV + PEEP did not prevent the development of hypoxia and was associated with increased pleural fluid formation. Death within 96 hours in the delayed group was the result of respiratory failure aggravated by bronchial cast formation despite vigorous pulmonary toilet. Early positive pressure ventilation with PEEP, preferably initiated immediately after the inhalation insult, significantly increases short-term survival and is associated with decreased tracheobronchial cast formation in this ovine model of severe smoke inhalation injury.

Animals↗

PubMed1991

Sequence requirements for the editing of apolipoprotein B mRNA.

Apolipoprotein (apo) B48 is produced in the mammalian intestine by a tissue-specific RNA-editing mechanism, which mediates a C to U conversion at position 6666 in apoB mRNA. This generates an inframe translation stop codon (UAA) in place of glutamine (CAA) at position 2153. To establish the sequences required for editing we have used an in vitro conversion assay to monitor the editing of synthetic RNAs by rat intestinal extracts. Transcripts containing 55 nucleotides (positions 6649-6703) or more of human apoB mRNA sequence were edited in vitro. Transcripts containing 42 nucleotides (positions 6648-6689) and 26 nucleotides (positions 6662-6687) were edited at 62 and 24% efficiency, respectively, of the 55-nucleotide sequence. To delineate the precise sequence requirements for editing, mutants were generated where 6-nucleotide sections of the 55-base region were changed to anti-sense sequence. Mutation of the 12-nucleotide region immediately downstream of C-6666 abolished editing, and mutation of 6-base sequences immediately 3' and 5' of this 12-nucleotide region significantly reduced editing. Having identified the key region of interest, a panel of 46 mutant RNAs carrying single base substitutions or deletions between nucleotide positions 6657 and 6685 was constructed. Mutagenesis in the sequence 5'-TGATCAGTATA-3' (positions 6671-6681) downstream of C-6666 had the most dramatic effect, since almost all mutations abolished or greatly reduced conversion in vitro. These results suggest that editing is a highly sequence-specific process. We propose that this downstream region is a recognition and/or binding site for the editing enzyme. A search for this sequence in other genes may help to reveal other RNAs that undergo editing.

Animals↗

PubMed1991

Characterization of the apolipoprotein B mRNA editing enzyme: no similarity to the proposed mechanism of RNA editing in kinetoplastid protozoa.

Intestinal apolipoprotein B mRNA is edited at nucleotide 6666 by a C to U transition resulting in a translational stop codon. The enzymatic properties of the editing activity were characterised in vitro using rat enterocyte cytosolic extract. The editing activity has no nucleotide or ion cofactor requirement. It shows substrate saturation with an apparent Km for the RNA substrate of 2.2 nM. The editing enzyme requires no lag period prior to catalysis, and does not assemble into a higher order complex on the RNA substrate. In crude cytosolic extract editing activity is completely abolished by treatment with micrococcal nuclease or RNAse A. Partially purified editing enzyme is no longer sensitive to nucleases, but is inhibited in a dose dependent manner by nuclease inactivated crude extract. The buoyant density of partially purified editing enzyme is 1.3 g/ml, that of pure protein. Therefore, the apolipoprotein B mRNA editing activity consists of a well defined enzyme with no RNA component. The nuclease sensitivity in crude cytosolic extract is explained by the generation of inhibitors for the editing enzyme. The editing of apo B mRNA has little similarity to complex mRNA processing events such as splicing and unlike editing in kinetoplastid protozoa does not utilise guide RNAs.

APOBEC-1 Deaminase↗

PubMed1991

An additional editing site is present in apolipoprotein B mRNA.

Human intestinal apolipoprotein (apo) B mRNA undergoes a C to U RNA editing at nucleotide 6666 to generate a translation stop at codon 2153, which defines the carboxy-terminal of apo B48. Here we show that two of eleven human intestinal cDNAs spanning residue 6666 were edited from a genomically-encoded C to a T at residue 6802 as well as at residue 6666. This additional editing converts Thr (ACA) codon 2198 to Ile (AUA). Synthetic RNA including the nucleotide 6802 was edited in vitro by intestinal extracts at 10-15% of the editing efficiency of nucleotide 6666. A sequence is identified as important for recognition by the editing activity. No secondary structural homology was identified between the two edited sites. No other sequence in the region between 6411 and 6893 nucleotides of apo B mRNA was found to be edited in vivo or in vitro. Apo B RNA editing extracts from intestine did not edit maize cytochrome oxidase II mRNA.

Apolipoproteins B↗

PubMed1991

Site-specific creation of uridine from cytidine in apolipoprotein B mRNA editing.

Human apolipoprotein (apo) B mRNA is edited in a tissue specific reaction, to convert glutamine codon 2153 (CAA) to a stop translation codon. The RNA editing product templates and hybridises as uridine, but the chemical nature of this reaction and the physical identity of the product are unknown. After editing in vitro of [32P] labelled RNA, we are able to demonstrate the production of uridine from cytidine; [alpha 32P] cytidine triphosphate incorporated into RNA gave rise to [32P] uridine monophosphate after editing in vitro, hydrolysis with nuclease P1 and thin layer chromatography using two separation systems. By cleaving the RNA into ribonuclease T1 fragments, we show that uridine is produced only at the authentic editing site and is produced in quantities that parallel an independent primer extension assay for editing. We conclude that apo B mRNA editing specifically creates a uridine from a cytidine. These observations are inconsistent with the incorporation of a uridine nucleotide by any polymerase, which would replace the alpha-phosphate and so rule out a model of endonucleolytic excision and repair as the mechanism for the production of uridine. Although transamination and transglycosylation remain to be formally excluded as reaction mechanisms our results argue strongly in favour of the apo B mRNA editing enzyme as a site-specific cytidine deaminase.

Apolipoproteins B↗

PubMed1990

Purification, properties and partial amino acid sequence of the blood-group-A-gene-associated alpha-3-N-acetylgalactosaminyltransferase from human gut mucosal tissue.

An alpha-3-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine from UDP-N-acetylgalactosamine to H-active structures to form A determinants was purified to homogeneity from human gut mucosal tissue of blood-group-A subjects. The mucosa was homogenized, then treated with Triton X-100, and the solubilized enzyme was purified by affinity chromatography on UDP-hexanolamine-agarose and octyl-Sepharose CL-4B. Enzyme activity was recovered in 44% yield with a specific activity of approx. 7 mumol/min per mg. The only effective acceptor substrates for the transferase were those containing a subterminal beta-galactosyl residue substituted at the O-2 position with L-fucose. The purified enzyme had a weak capacity to transfer D-galactose from UDP-D-galactose to similar acceptors to make blood-group-B determinants. H.p.l.c. and SDS/PAGE analysis indicated an Mr of 40,000 for the purified enzyme. For the first time a partial amino acid sequence Xaa-Ser-Leu-Pro-Arg-Met-Val-Tyr-Pro-Gln-Ile-Ser?-Val-Leu was obtained for the N-terminal region of the soluble alpha-3-N-acetylgalactosaminyltransferase.

ABO Blood-Group System↗

PubMed1990

A comparison of effects of thermal injury and smoke inhalation on bacterial translocation.

Thermal injury as well as smoke inhalation injury results in serious morbidity and high mortality. In a chronic ovine model, we studied the development of bacterial translocation to the mesenteric lymph node, liver, spleen, kidney, and lung following: 1) sham injury (N = 6), 2) cutaneous thermal injury (N = 5), 3) cotton smoke inhalation injury (N = 4), 4) combined thermal injury and smoke inhalation injury (N = 7). Cardiac output, mean arterial pressure, and plasma protein concentration were maintained within 10% of preinjury values. Urine output was maintained above 1 ml/kg/hour with fluid and plasma resuscitation. A wide-beam ultrasonic flow probe was chronically implanted to allow serial measurement of cephalic mesenteric arterial blood flow throughout the 48-hour experimental period. Sheep were sacrificed 48 hours following injury for quantitative organ culture of mesenteric lymph node, liver, spleen, kidney, and lung. Measurements of mesenteric blood flow demonstrated a decrease to 48 +/- 8%, 80 +/- 5%, and 64 +/- 9% of preinjury levels in sheep receiving thermal injury, smoke inhalation injury, and combination injury, respectively. The sham animals maintained mesenteric blood flow at 102 +/- 7% of control levels. Thermal injury, as well as combination thermal and smoke inhalation injury, resulted in higher levels of translocation than smoke inhalation injury alone.

Animals↗

PubMed1988

Purification, properties and cation activation of galactosyltransferase from lactating-rat mammary Golgi membranes.

Galactosyltransferase was purified from Golgi membranes of lactating-rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties. The enzyme occurred in both monomeric (43-46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N-terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro-Pro-Pro-Pro sequence. Kinetic studies demonstrated a high-affinity Mn2+-binding site (1) essential for activity, and a low-affinity Mn2+-binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase. It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.

Amino Acid Sequence↗

PubMed1986

Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins.

Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.

Animals↗

PubMed1986

Modulation of activity of human alkaline phosphatases by Mg2+ and thiol compounds.

Interaction of purified human liver and placental alkaline phosphatases (orthophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1) with sulfhydryl groups, sulfhydryl reagents, and Mg2+ were studied. L-Cysteine (0.1 mmol/l) or Mg2+ activated the liver enzyme 4-5-fold and the placental enzyme 2-3-fold, with optimal pH 7.5-8.0; these activations were not additive. L-Cysteine (2 mmol/l) inhibited both enzymes maximally at pH greater than 9.0; phosphate protected the enzymes. S-Methylcysteine had little effect, with or without Mg2+. Inhibition by sulfur-containing compounds paralleled their ability to bind Zn2+. Fluoresceine mercury acetate (specific for sulfhydryl groups) inhibited the isoenzymes, whereas iodoacetic acid, iodoacetamide, dithionitrobenzoic acid, and p-chloromercuribenzoate had little effect. The inhibition was reversed by L-cysteine and only slightly protected by inorganic phosphate. Thus, there are two sites on human liver and placental alkaline phosphatase that interact with L-cysteine; a Mg2+-binding site, which results in activation, and a site that involves one or both of the bound Zn2+ ions and results in inactivation. Both enzymes have a protected essential thiol group.

Alkaline Phosphatase↗

PubMed1985

Reactive thiol groups in rat liver acid phosphatase.

Dimeric rat liver acid phosphatase P1 of Mr 92,000 is inactivated by p-chloromercuribenzoate and fluorescein mercuriacetate (FMA). The enzyme is protected against the mercurials by the substrate analogue Pi. The reaction with FMA is accompanied by changes in absorbance at 495 nm and in fluorescence emission at 520 nm that are characteristic of reaction of this compound with thiol groups. Titration of P1 with FMA monitored by spectrophotometry or by fluorimetry indicated that equivalence is reached at an FMA/P1 ratio of 3. Since FMA can act as a bifunctional reagent, it is likely that P1 contains either 3 or 6 reactive thiol groups per molecule. Analysis of FMA inactivation/modification data by a statistical method suggests that of 6 reactive thiol groups, 2 are essential so that there are probably 3 thiol groups per subunit, one of which is located at the active site. If the total thiol number is 3, analysis suggests 1 essential thiol per subunit.

Acid Phosphatase↗